Antisense modulation of raidd expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of RAIDD. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding RAIDD. Methods of using these compounds for modulation of RAIDD expression and for treatment of diseases associated with expression of RAIDD are provided.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of RAIDD. In particular, this invention relates tocompounds, particularly oligonucleotides, specifically hybridizable withnucleic acids encoding RAIDD. Such compounds have been shown to modulatethe expression of RAIDD.

BACKGROUND OF THE INVENTION

Apoptosis, or programmed cell death, is a naturally occurring processthat has been strongly conserved during evolution to preventuncontrolled cell proliferation. This form of cell suicide plays acrucial role in ensuring the development and maintenance ofmulticellular organisms by eliminating superfluous or unwanted cells.However, if this process goes awry becoming overstimulated, cell lossand degenerative disorders including neurological disorders such asAlzheimers, Parkinsons, ALS, retinitis pigmentosa and blood celldisorders can result. Stimuli which can trigger apoptosis include growthfactors such as tumor necrosis factor (TNF), Fas and transforming growthfactor beta (TGFβ), neurotransmitters, growth factor withdrawal, loss ofextracellular matrix attachment and extreme fluctuations inintracellular calcium levels (Afford and Randhawa, Mol. Pathol., 2000,53, 55-63).

Alternatively, insufficient apoptosis, triggered by growth factors,extracellular matrix changes, CD40 ligand, viral gene products neutralamino acids, zinc, estrogen and androgens, can contribute to thedevelopment of cancer, autoimmune disorders and viral infections (Affordand Randhawa, Mol. Pathol., 2000, 53, 55-63).

Although several stimuli can induce apoptosis, little is known about theintermediate signaling events, including inhibition, that connect theapoptotic signal to a common cell death pathway conserved across manyspecies. Recently, major advances have been made in understanding thesignaling pathways mediated by the tumor necrosis factor receptor (TNFR)family which signals apoptosis.

Two cell surface cytokine receptors of the TNFR family, TNFR-1 and CD95(Fas/APO-1), act as death receptors and a number of binding proteinshave been identified which mediate apoptosis through these receptors.The pathways leading from these receptors involve a proteolytic cascadeorchestrated by a family of enzymes known as caspases (Thornberry,British Medical Bulletin, 1997, 53, 478-490).

Caspases activate apoptosis through proteolytic events triggered by oneof several described mechanisms; including ligand binding to the cellsurface death receptors of either the TNF or NGF receptor families,changes in mitochondrial integrity or chemical induction (Thornberry,British Medical Bulletin, 1997, 53, 478-490).

To a large extent, caspase, and consequently apoptotic, signaling ismediated by protein-protein interactions between caspases and theiradapter molecules (Cohen, Biochemistry Journal, 1997, 326, 1-16). Theseinteractions have been localized to three independently foldinginteraction domains known as the death domain (DD), the death effectordomain (DED) and the caspase recruitment domain (CARD) (Hofmann, Cell.Mol. Life Sci., 1999, 55, 1113-1128).

RAIDD (also known as CRADD for CASP2 and RIPK1 domain containing Adaptorwith Death Domain) is an adaptor molecule which contains both DD andCARD domains. It has been shown to interact with caspase-2 via the CARDdomains found in each and with RIP, a serine/threonine kinase, via therespective DD domains. Both caspase-2 and RIP subsequently associatewith tumor necrosis factor receptor 1 (TNFR-1) through theseinteractions with RAIDD (Ahmad et al., Cancer Res., 1997, 57, 615-619;Duan and Dixit, Nature, 1997, 385, 86-89).

Characterization of the localization of the RAIDD protein revealed thatthe mRNA is found in various tissues and that expression occurs in mostadult tissues and in fetal liver. The highest expression was observed inthe thymus, testes and fetal liver (Ahmad et al., Cancer Res., 1997, 57,615-619). The protein is found predominantly in the cytoplasm and tosome extent in the nucleus (Shearwin-whyatt et al., Cell Death Differ.,2000, 7, 155-165). This localization was not found to be affected byTNF-treatment in HeLa or 293T cells.

Overexpression of RAIDD has been shown to induce apoptosis in mammaliancells including human embryonic kidney cells and a breast carcinoma cellline, MCF-7 (Ahmad et al., Cancer Res., 1997, 57, 615-619; Duan andDixit, Nature, 1997, 385, 86-89). Overexpression of the CARD domain ofRAIDD has been shown to induce the formation of filamentous structuresdue to CARD-mediated oligomerization (Shearwin-Whyatt et al., Cell DeathDiffer., 2000, 7, 155-165). This type of complex formation has beenpreviously shown to occur with the overexpression of other deathdomains. Consequently, the pharmacological modulation of RAIDD activityand/or expression is therefore believed to be an appropriate point oftherapeutic intervention in pathological conditions involving theaberrant modulation of apoptosis.

Recently, the human and mouse RAIDD genes have been mapped tochromosomes 12 and 10, respectively (Horvat and Medrano, Genomics, 1998,54, 159-164). This region contains the putative site for the hg (highgrowth) gene of the mouse. The hg gene produces an increase in weightgain of up to 50% in homozygous individuals with no concomittantincrease in obesity. Using exon trapping studies, Horvat et al. suggestthat RAIDD might actually encode the hg gene of the mouse (Horvat andMedrano, Genomics, 1998, 54, 159-164). The results of these studiessuggest that RAIDD may be an attractive therapeutic target for growth ormetabolic disorders.

Currently, there are no known therapeutic agents which effectivelyinhibit the synthesis of RAIDD and to date, investigative strategiesaimed at modulating RAIDD function have involved the use of antibodiesand truncated forms of the protein containing the death domains.Consequently, there remains a long felt need for agents capable ofeffectively inhibiting RAIDD function.

Antisense technology is emerging as an effective means for reducing theexpression of specific gene products and may therefore prove to beuniquely useful in a number of therapeutic, diagnostic, and researchapplications for the modulation of RAIDD expression.

The present invention provides compositions and methods for modulatingRAIDD expression.

SUMMARY OF THE INVENTION

The present invention is directed to compounds, particularly antisenseoligonucleotides, which are targeted to a nucleic acid encoding RAIDD,and which modulate the expression of RAIDD. Pharmaceutical and othercompositions comprising the compounds of the invention are alsoprovided. Further provided are methods of modulating the expression ofRAIDD in cells or tissues comprising contacting said cells or tissueswith one or more of the antisense compounds or compositions of theinvention. Further provided are methods of treating an animal,particularly a human, suspected of having or being prone to a disease orcondition associated with expression of RAIDD by administering atherapeutically or prophylactically effective amount of one or more ofthe antisense compounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention employs oligomeric compounds, particularlyantisense oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding RAIDD, ultimately modulating the amountof RAIDD produced. This is accomplished by providing antisense compoundswhich specifically hybridize with one or more nucleic acids encodingRAIDD. As used herein, the terms “target nucleic acid” and “nucleic acidencoding RAIDD” encompass DNA encoding RAIDD, RNA (including pre-mRNAand mRNA) transcribed from such DNA, and also cDNA derived from suchRNA. The specific hybridization of an oligomeric compound with itstarget nucleic acid interferes with the normal function of the nucleicacid. This modulation of function of a target nucleic acid by compoundswhich specifically hybridize to it is generally referred to as“antisense”. The functions of DNA to be interfered with includereplication and transcription. The functions of RNA to be interferedwith include all vital functions such as, for example, translocation ofthe RNA to the site of protein translation, translation of protein fromthe RNA, splicing of the RNA to yield one or more mRNA species, andcatalytic activity which may be engaged in or facilitated by the RNA.The overall effect of such interference with target nucleic acidfunction is modulation of the expression of RAIDD. In the context of thepresent invention, “modulation” means either an increase (stimulation)or a decrease (inhibition) in the expression of a gene. In the contextof the present invention, inhibition is the preferred form of modulationof gene expression and mRNA is a preferred target.

It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding RAIDD. The targeting process also includesdetermination of a site or sites within this gene for the antisenseinteraction to occur such that the desired effect, e.g., detection ormodulation of expression of the protein, will result. Within the contextof the present invention, a preferred intragenic site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since, as is known in the art, thetranslation initiation codon is typically 5′-AUG (in transcribed mRNAmolecules; 5′-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the “AUG codon,” the “startcodon” or the “AUG start codon”. A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, theterms “translation initiation codon” and “start codon” can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, “start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of an mRNA molecule transcribed from a geneencoding RAIDD, regardless of the sequence(s) of such codons.

It is also known in the art that a translation termination codon (or“stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA,5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAGand 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Other target regions include the 5′ untranslatedregion (5′UTR), known in the art to refer to the portion of an mRNA inthe 5′ direction from the translation initiation codon, and thusincluding nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. mRNA splice sites, i.e., intron-exonjunctions, may also be preferred target regions, and are particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular mRNA splice product isimplicated in disease. Aberrant fusion junctions due to rearrangementsor deletions are also preferred targets. It has also been found thatintrons can also be effective, and therefore preferred, target regionsfor antisense compounds targeted, for example, to DNA or pre-mRNA.

Once one or more target sites have been identified, oligonucleotides arechosen which are sufficiently complementary to the target, i.e.,hybridize sufficiently well and with sufficient specificity, to give thedesired effect.

In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementary orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a loss ofutility, and there is a sufficient degree of complementary to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed.

Antisense and other compounds of the invention which hybridize to thetarget and inhibit expression of the target are identified throughexperimentation, and the sequences of these compounds are hereinbelowidentified as preferred embodiments of the invention. The target sitesto which these preferred sequences are complementary are hereinbelowreferred to as “active sites” and are therefore preferred sites fortargeting. Therefore another embodiment of the invention encompassescompounds which hybridize to these active sites.

Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense oligonucleotideshave been employed as therapeutic moieties in the treatment of diseasestates in animals and man. Antisense oligonucleotide drugs, includingribozymes, have been safely and effectively administered to humans andnumerous clinical trials are presently underway. It is thus establishedthat oligonucleotides can be useful therapeutic modalities that can beconfigured to be useful in treatment regimes for treatment of cells,tissues and animals, especially humans.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA) or mimetics thereof. This term includes oligonucleotidescomposed of naturally-occurring nucleobases, sugars and covalentinternucleoside (backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

While antisense oligonucleotides are a preferred form of antisensecompound, the present invention comprehends other oligomeric antisensecompounds, including but not limited to oligonucleotide mimetics such asare described below. The antisense compounds in accordance with thisinvention preferably comprise from about 8 to about 50 nucleobases (i.e.from about 8 to about 50 linked nucleosides). Particularly preferredantisense compounds are antisense oligonucleotides, even more preferablythose comprising from about 12 to about 30 nucleobases. Antisensecompounds include ribozymes, external guide sequence (EGS)oligonucleotides (oligozymes), and other short catalytic RNAs orcatalytic oligonucleotides which hybridize to the target nucleic acidand modulate its expression.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn the respective ends of this linear polymericstructure can be further joined to form a circular structure, however,open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates, 5′-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidatesincluding 3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs of these, and thosehaving inverted polarity wherein one or more internucleotide linkages isa 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotideshaving inverted polarity comprise a single 3′ to 3′ linkage at the3′-most internucleotide linkage i.e. a single inverted nucleosideresidue which may be abasic (the nucleobase is missing or has a hydroxylgroup in place thereof). Various salts, mixed salts and free acid formsare also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage, i.e., the backbone, of the nucleotide units arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

Most preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S—or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10.Other preferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

A further prefered modification includes Locked Nucleic Acids (LNAs) inwhich the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of thesugar ring thereby forming a bicyclic sugar moiety. The linkage ispreferably a methelyne (—CH₂—)_(n) group bridging the 2′ oxygen atom andthe 3′ or 4′ carbon atom wherein n is 1 or 2. LNAs and preparationthereof are described in WO 98/39352 and WO 99/14226.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′—OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further modified nucleobases include tricyclicpyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b ]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the oligomeric compoundsof the invention. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. Nos. 3,687,808, as well as U.S. Pat. Nos. 4,845,205;5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588;6,005,096; and 5,681,941, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, which is commonly owned with theinstant application and also herein incorporated by reference.

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. The compounds of the invention caninclude conjugate groups covalently bound to functional groups such asprimary or secondary hydroxyl groups. Conjugate groups of the inventioninclude intercalators, reporter molecules, polyamines, polyamides,polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugates groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improve oligomeruptake, enhance oligomer resistance to degradation, and/or strengthensequence-specific hybridization with RNA. Groups that enhance thepharmacokinetic properties, in the context of this invention, includegroups that improve oligomer uptake, distribution, metabolism orexcretion. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992 theentire disclosure of which is incorporated herein by reference.Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937. Oligonucleotides of the invention mayalso be conjugated to active drug substances, for example, aspirin,warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain ofwhich are commonly owned with the instant application, and each of whichis herein incorporated by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

The antisense compounds of the invention are synthesized in vitro and donot include antisense compositions of biological origin, or geneticvector constructs designed to direct the in vivo synthesis of antisensemolecules. The compounds of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto prodrugs and pharmaceutically acceptable salts of the compounds ofthe invention, pharmaceutically acceptable salts of such prodrugs, andother bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphates] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotides, preferred examples of pharmaceutically acceptablesalts include but are not limited to (a) salts formed with cations suchas sodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

The antisense compounds of the present invention can be utilized fordiagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of RAIDD is treated by administering antisense compounds inaccordance with this invention. The compounds of the invention can beutilized in pharmaceutical compositions by adding an effective amount ofan antisense compound to a suitable pharmaceutically acceptable diluentor carrier. Use of the antisense compounds and methods of the inventionmay also be useful prophylactically, e.g., to prevent or delayinfection, inflammation or tumor formation, for example.

The antisense compounds of the invention are useful for research anddiagnostics, because these compounds hybridize to nucleic acids encodingRAIDD, enabling sandwich and other assays to easily be constructed toexploit this fact. Hybridization of the antisense oligonucleotides ofthe invention with a nucleic acid encoding RAIDD can be detected bymeans known in the art. Such means may include conjugation of an enzymeto the oligonucleotide, radiolabelling of the oligonucleotide or anyother suitable detection means. Kits using such detection means fordetecting the level of RAIDD in a sample may also be prepared.

The present invention also includes pharmaceutical compositions andformulations which include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Oligonucleotideswith at least one 2′-O-methoxyethyl modification are believed to beparticularly useful for oral administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Preferred topical formulations include those inwhich the oligonucleotides of the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Preferredlipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidylglycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). Oligonucleotides of theinvention may be encapsulated within liposomes or may form complexesthereto, in particular to cationic liposomes. Alternatively,oligonucleotides may be complexed to lipids, in particular to cationiclipids. Preferred fatty acids and esters include but are not limitedarachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylicacid, capric acid, myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin,glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine,an acylcholine, or a C₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM),monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.Topical formulations are described in detail in U.S. patent applicationSer. No. 09/315,298 filed on May 20, 1999 which is incorporated hereinby reference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Prefered bileacids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate. Preferedfatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium). Also prefered are combinations of penetrationenhancers, for example, fatty acids/salts in combination with bileacids/salts. A particularly prefered combination is the sodium salt oflauric acid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.Oligonucleotides of the invention may be delivered orally in granularform including sprayed dried particles, or complexed to form micro ornanoparticles. Oligonucleotide complexing agents include poly-aminoacids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Particularly preferred complexing agentsinclude chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine,polyornithine, polyspermines, protamine, polyvinylpyridine,polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor oligonucleotides and their preparation are described in detail inU.S. application Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No.09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23,1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298(filed May 20, 1999) each of which is incorporated herein by referencein their entirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogenous systems of one liquiddispersed in another in the form of droplets usually exceeding 0.1 μm indiameter. (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p.245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335;Higuchi et al., in Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systemscomprising of two immiscible liquid phases intimately mixed anddispersed with each other. In general, emulsions may be eitherwater-in-oil (w/o) or of the oil-in-water (o/w) variety. When an aqueousphase is finely divided into and dispersed as minute droplets into abulk oily phase the resulting composition is called a water-in-oil (w/o)emulsion. Alternatively, when an oily phase is finely divided into anddispersed as minute droplets into a bulk aqueous phase the resultingcomposition is called an oil-in-water (o/w) emulsion. Emulsions maycontain additional components in addition to the dispersed phases andthe active drug which may be present as a solution in either the aqueousphase, oily phase or itself as a separate phase. Pharmaceuticalexcipients such as emulsifiers, stabilizers, dyes, and anti-oxidants mayalso be present in emulsions as needed. Pharmaceutical emulsions mayalso be multiple emulsions that are comprised of more than two phasessuch as, for example, in the case of oil-in-water-in-oil (o/w/o) andwater-in-oil-in-water (w/o/w) emulsions. Such complex formulations oftenprovide certain advantages that simple binary emulsions do not. Multipleemulsions in which individual oil droplets of an o/w emulsion enclosesmall water droplets constitute a w/o/w emulsion. Likewise a system ofoil droplets enclosed in globules of water stabilized in an oilycontinuous provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of reasons of ease of formulation, efficacyfrom an absorption and bioavailability standpoint. (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C₁₀glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsionscompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Liposomes

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes. As the mergingof the liposome and cell progresses, the liposomal contents are emptiedinto the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g. as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(ml), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(Ml), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(Ml) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A limited number of liposomes comprising nucleic acids are known in theart. WO 96/40062 to Thierry et al. discloses methods for encapsulatinghigh molecular weight nucleic acids in liposomes. U.S. Pat. No.5,264,221 to Tagawa et al. discloses protein-bonded liposomes andasserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p.92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

Surfactants: In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of oligonucleotides through the mucosais enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. The bile salts of the inventioninclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with thepresent invention, can be defined as compounds that remove metallic ionsfrom solution by forming complexes therewith, with the result thatabsorption of oligonucleotides through the mucosa is enhanced. Withregards to their use as penetration enhancers in the present invention,chelating agents have the added advantage of also serving as DNaseinhibitors, as most characterized DNA nucleases require a divalent metalion for catalysis and are thus inhibited by chelating agents (Jarrett,J. Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption ofoligonucleotides through the alimentary mucosa (Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This classof penetration enhancers include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92);and non-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

Agents that enhance uptake of oligonucleotides at the cellular level mayalso be added to the pharmaceutical and other compositions of thepresent invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate oligonucleotide in hepatic tissue can be reduced whenit is coadministered with polyinosinic acid, dextran sulfate,polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonicacid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura etal., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

Pharmaceutically acceptable organic or inorganic excipient suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Certain embodiments of the invention provide pharmaceutical compositionscontaining (a) one or more antisense compounds and (b) one or more otherchemotherapeutic agents which function by a non-antisense mechanism.Examples of such chemotherapeutic agents include but are not limited todaunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin,idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosinearabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions of the invention. See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 2499-2506 and 46-49, respectively). Other non-antisensechemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Numerous examples of antisensecompounds are known in the art. Two or more combined compounds may beused together or sequentially.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1 Nucleoside Phosphoramidites for OligonucleotideSynthesis Deoxy and 2′-alkoxy Amidites

2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropyl phosphoramidites werepurchased from commercial sources (e.g. Chemgenes, Needham Ma. or GlenResearch, Inc. Sterling Va.). Other 2′-O-alkoxy substituted nucleosideamidites are prepared as described in U.S. Pat. No. 5,506,351, hereinincorporated by reference. For oligonucleotides synthesized using2′-alkoxy amidites, the standard cycle for unmodified oligonucleotideswas utilized, except the wait step after pulse delivery of tetrazole andbase was increased to 360 seconds.

Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me—C)nucleotides were synthesized according to published methods [Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203] using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham Ma.).

2′-Fluoro Amidites 2′-Fluorodeoxyadenosine Amidites

2′-fluoro oligonucleotides were synthesized as described previously[Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and U.S. Pat. No.5,670,633, herein incorporated by reference. Briefly, the protectednucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesizedutilizing commercially available 9-beta-D-arabinofuranosyladenine asstarting material and by modifying literature procedures whereby the2′-alpha-fluoro atom is introduced by a S_(N)2-displacement of a2′-beta-trityl group. Thus N6-benzoyl-9-beta-D-arabinofuranosyladeninewas selectively protected in moderate yield as the3′,5′-ditetrahydropyranyl (THP) intermediate. Deprotection of the THPand N6-benzoyl groups was accomplished using standard methodologies andstandard methods were used to obtain the 5′-dimethoxytrityl-(DMT) and5′-DMT-3′-phosphoramidite intermediates.

2′-Fluorodeoxyguanosine

The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplished usingtetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

2′-Fluorouridine

Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′ phosphoramidites.

2′-Fluorodeoxycytidine

2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

2′-O-(2-Methoxyethyl) Modified Amidites

2′-O-Methoxyethyl-substituted nucleoside amidites are prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g, 0.420 M)and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). Themixture was heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution was concentrated under reduced pressure. Theresulting syrup was poured into diethylether (2.5 L), with stirring. Theproduct formed a gum. The ether was decanted and the residue wasdissolved in a minimum amount of methanol (ca. 400 mL). The solution waspoured into fresh ether (2.5 L) to yield a stiff gum. The ether wasdecanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for24 h) to give a solid that was crushed to a light tan powder (57 g, 85%crude yield). The NMR spectrum was consistent with the structure,contaminated with phenol as its sodium salt (ca. 5%). The material wasused as is for further reactions (or it can be purified further bycolumn chromatography using a gradient of methanol in ethyl acetate(10-25%) to give a white solid, mp 222-4° C.).

2′-O-Methoxyethyl-5-methyluridine

2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate(231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 Lstainless steel pressure vessel and placed in a pre-heated oil bath at160° C. After heating for 48 hours at 155-160° C., the vessel was openedand the solution evaporated to dryness and triturated with MeOH (200mL). The residue was suspended in hot acetone (1 L). The insoluble saltswere filtered, washed with acetone (150 mL) and the filtrate evaporated.The residue (280 g) was dissolved in CH₃CN (600 mL) and evaporated. Asilica gel column (3 kg) was packed in CH₂Cl₂/acetone/MeOH (20:5:3)containing 0.5% Et₃NH. The residue was dissolved in CH₂Cl₂ (250 mL) andadsorbed onto silica (150 g) prior to loading onto the column. Theproduct was eluted with the packing solvent to give 160 g (63%) ofproduct. Additional material was obtained by reworking impure fractions.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporatedwith pyridine (250 mL) and the dried residue dissolved in pyridine (1.3L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the mixture stirred at room temperature for one hour. A secondaliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and thereaction stirred for an additional one hour. Methanol (170 mL) was thenadded to stop the reaction. HPLC showed the presence of approximately70% product. The solvent was evaporated and triturated with CH₃CN (200mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phasewas dried over Na₂SO₄, filtered and evaporated. 275 g of residue wasobtained. The residue was purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/hexane/acetone (5:5:1) containing 0.5% Et₃NH. Thepure fractions were evaporated to give 164 g of product. Approximately20 g additional was obtained from the impure fractions to give a totalyield of 183 g (57%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M),DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) werecombined and stirred at room temperature for 24 hours. The reaction wasmonitored by TLC by first quenching the TLC sample with the addition ofMeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the latter solution. The resulting reaction mixturewas stored overnight in a cold room. Salts were filtered from thereaction mixture and the solution was evaporated. The residue wasdissolved in EtOAc (1 L) and the insoluble solids were removed byfiltration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mLof saturated NaCl, dried over sodium sulfate and evaporated. The residuewas triturated with EtOAc to give the title compound.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (TLC showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M)was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M)was added with stirring. After stirring for 3 hours, TLC showed thereaction to be approximately 95% complete. The solvent was evaporatedand the residue azeotroped with MeOH (200 mL). The residue was dissolvedin CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) andsaturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give aresidue (96 g). The residue was chromatographed on a 1.5 kg silicacolumn using EtOAc/hexane (1:1) containing 0.5% Et₃NH as the elutingsolvent. The pure product fractions were evaporated to give 90 g (90%)of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (TLC showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites2′-(Dimethylaminooxyethoxy) nucleoside amidites

2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known in the artas 2′-O-(dimethylaminooxyethyl) nucleoside amidites] are prepared asdescribed in the following paragraphs. Adenosine, cytidine and guanosinenucleoside amidites are prepared similarly to the thymidine(5-methyluridine) except the exocyclic amines are protected with abenzoyl moiety in the case of adenosine and cytidine and with isobutyrylin the case of guanosine.

5′-O-tert-Butyldiphenylsilyl-O²-2-anhydro-5-methyluridine

O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g,0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) weredissolved in dry pyridine (500 ml) at ambient temperature under an argonatmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane(125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. Thereaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22,ethyl acetate) indicated a complete reaction. The solution wasconcentrated under reduced pressure to a thick oil. This was partitionedbetween dichloromethane (1 L) and saturated sodium bicarbonate (2×1 L)and brine (1 L). The organic layer was dried over sodium sulfate andconcentrated under reduced pressure to a thick oil. The oil wasdissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600 mL) andthe solution was cooled to −10° C. The resulting crystalline product wascollected by filtration, washed with ethyl ether (3×200 mL) and dried(40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLC and NMRwere consistent with pure product.

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

In a 2 L stainless steel, unstirred pressure reactor was added borane intetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and withmanual stirring, ethylene glycol (350 mL, excess) was added cautiouslyat first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure<100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct.

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried overP₂O₅ under high vacuum for two days at 40° C. The reaction mixture wasflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete, thereaction was stirred for 4 hrs. By that time TLC showed the completionof the reaction (ethylacetate:hexane, 60:40). The solvent was evaporatedin vacuum. Residue obtained was placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%).

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 h the mixture was filtered, the filtrate was washed with icecold CH₂Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂SO₄. The solution was concentrated to get2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was addedand the resulting mixture was strirred for 1 h. Solvent was removedunder vacuum; residue chromatographed to get5′-O-tert-butyldiphenylsilyl-2′O-[(2-formadoximinooxy)ethyl]-5-methyluridineas white foam (1.95 g, 78%).

5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) was added to this solution at 10° C. under inertatmosphere. The reaction mixture was stirred for 10 minutes at 10° C.After that the reaction vessel was removed from the ice bath and stirredat room temperature for 2 h, the reaction monitored by TLC (5% MeOH inCH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extractedwith ethyl acetate (2×20 mL). Ethyl acetate phase was dried overanhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in asolution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL,3.37 mmol) was added and the reaction mixture was stirred at roomtemperature for 10 minutes. Reaction mixture cooled to 10° C. in an icebath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reactionmixture stirred at 10° C. for 10 minutes. After 10 minutes, the reactionmixture was removed from the ice bath and stirred at room temperaturefor 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was addedand extracted with ethyl acetate (2×25 mL). Ethyl acetate layer wasdried over anhydrous Na₂SO₄ and evaporated to dryness. The residueobtained was purified by flash column chromatography and eluted with 5%MeOH in CH₂Cl₂ to get5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%).

2′-O-(dimethylaminooxyethyl)-5-methyluridine

Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dryTHF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). Thismixture of triethylamine-2HF was then added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂Cl₂). Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) wasdried over P₂O₅ under high vacuum overnight at 40° C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get5′-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67mmol) was co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and driedover P₂O₅ under high vacuum overnight at 40° C. Then the reactionmixture was dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated,then the residue was dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrousNa₂SO₄ and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

2′-(Aminooxyethoxy) nucleoside amidites

2′-(Aminooxyethoxy) nucleoside amidites [also known in the art as2′-O-(aminooxyethyl) nucleoside amidites] are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

The 2′-O-aminooxyethyl guanosine analog may be obtained by selective2′-O-alkylation of diaminopurine riboside. Multigram quantities ofdiaminopurine riboside may be purchased from Schering AG (Berlin) toprovide 2′-O-(2-ethylacetyl) diaminopurine riboside along with a minoramount of the 3′-O-isomer. 2′-O-(2-ethylacetyl) diaminopurine ribosidemay be resolved and converted to 2′-O-(2-ethylacetyl)guanosine bytreatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D.,Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection proceduresshould afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosineand2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside mayphosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites

2′-dimethylaminoethoxyethoxy nucleoside amidites (also known in the artas 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, or2′-DMAEOE nucleoside amidites) are prepared as follows. Other nucleosideamidites are prepared similarly.

2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine

2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) is slowlyadded to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10 mmol)with stirring in a 100 mL bomb. Hydrogen gas evolves as the soliddissolves. O²-,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodiumbicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oilbath and heated to 155° C. for 26 hours. The bomb is cooled to roomtemperature and opened. The crude solution is concentrated and theresidue partitioned between water (200 mL) and hexanes (200 mL). Theexcess phenol is extracted into the hexane layer. The aqueous layer isextracted with ethyl acetate (3×200 mL) and the combined organic layersare washed once with water, dried over anhydrous sodium sulfate andconcentrated. The residue is columned on silica gel usingmethanol/methylene chloride 1:20 (which has 2% triethylamine) as theeluent. As the column fractions are concentrated a colorless solid formswhich is collected to give the title compound as a white solid.

5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy) ethyl)]-5-methyluridine

To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reactionmixture is poured into water (200 mL) and extracted with CH₂Cl₂ (2×200mL). The combined CH₂Cl₂ layers are washed with saturated NaHCO₃solution, followed by saturated NaCl solution and dried over anhydroussodium sulfate. Evaporation of the solvent followed by silica gelchromatography using MeOH:CH₂Cl₂:Et₃N (20:1, v/v, with 1% triethylamine)gives the title compound.

5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

Diisopropylaminotetrazolide (0.6 g) and 2-cyanoethoxy-N,N-diisopropylphosphoramidite (1.1 mL, 2 eq.) are added to a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture is stirred overnight and the solventevaporated. The resulting residue is purified by silica gel flash columnchromatography with ethyl acetate as the eluent to give the titlecompound.

Example 2

Oligonucleotide Synthesis

Unsubstituted and substituted phosphodiester (P═O) oligonucleotides aresynthesized on an automated DNA synthesizer (Applied Biosystems model380B) using standard phosphoramidite chemistry with oxidation by iodine.

Phosphorothioates (P═S) are synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 h), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution.

Phosphinate oligonucleotides are prepared as described in U.S. Pat. No.5,508,270, herein incorporated by reference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,610,289 or U.S. Pat. No. 5,625,050, hereinincorporated by reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporated byreference.

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 3

Oligonucleoside Synthesis

Methylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethyl-hydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 4

PNA Synthesis

Peptide nucleic acids (PNAs) are prepared in accordance with any of thevarious procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5

Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]--[2′-deoxy]--[2′-O-Me ] Chimeric PhosphorothioateOligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 380B, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by increasing the wait stepafter the delivery of tetrazole and base to 600 s repeated four timesfor RNA and twice for 2′-O-methyl. The fully protected oligonucleotideis cleaved from the support and the phosphate group is deprotected in3:1 ammonia/ethanol at room temperature overnight then lyophilized todryness. Treatment in methanolic ammonia for 24 hrs at room temperatureis then done to deprotect all bases and sample was again lyophilized todryness. The pellet is resuspended in 1M TBAF in THF for 24 hrs at roomtemperature to deprotect the 2′ positions. The reaction is then quenchedwith 1M TEAA and the sample is then reduced to ½ volume by rotovacbefore being desalted on a G25 size exclusion column. The oligorecovered is then analyzed spectrophotometrically for yield and forpurity by capillary electrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]--[2′-deoxy]--[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[2′-(2-methoxyethyl)]--[2′-deoxy]--[-2′-O-(methoxy-ethyl)] chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]--[2′-deoxyPhosphorothioate]--[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[2′-O-(2-methoxyethyl phosphodiester]--[2′-deoxyphosphorothioate]--[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

Oligonucleotide Isolation

After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides were analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85% fulllength material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹P nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7

Oligonucleotide Synthesis—96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a standard 96 well format. Phosphodiesterinternucleotide linkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected beta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8

Oligonucleotide Analysis—96 Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9

Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing 5 cell types are provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, Ribonucleaseprotection assays, or RT-PCR.

T-24 Cells

The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10%fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.),penicillin 100 units per mL, and streptomycin 100 micrograms per mL(Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were seeded into 96-well plates (Falcon-Primaria#3872) at a density of 7000 cells/well for use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 Cells

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence.

NHDF Cells

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK Cells

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville Md). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

b.END Cells

The mouse brain endothelial cell line b.END was obtained from Dr. WernerRisau at the Max Plank Instititute (Bad Nauheim, Germany). b.END cellswere routinely cultured in DMEM, high glucose (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 3000cells/well for use in RT-PCR analysis.

For Northern blotting or other analyses, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

Treatment with Antisense Compounds

When cells reached 80% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and thentreated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™(Gibco BRL) and the desired concentration of oligonucleotide. After 4-7hours of treatment, the medium was replaced with fresh medium. Cellswere harvested 16-24 hours after oligonucleotide treatment.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG,SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown inbold) with a phosphorothioate backbone which is targeted to human H-ras.For mouse or rat cells the positive control oligonucleotide is ISIS15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer(2′-O-methoxyethyls shown in bold) with a phosphorothioate backbonewhich is targeted to both mouse and rat c-raf. The concentration ofpositive control oligonucleotide that results in 80% inhibition ofc-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is thenutilized as the screening concentration for new oligonucleotides insubsequent experiments for that cell line. If 80% inhibition is notachieved, the lowest concentration of positive control oligonucleotidethat results in 60% inhibition of H-ras or c-raf MRNA is then utilizedas the oligonucleotide screening concentration in subsequent experimentsfor that cell line. If 60% inhibition is not achieved, that particularcell line is deemed as unsuitable for oligonucleotide transfectionexperiments.

Example 10

Analysis of Oligonucleotide Inhibition of RAIDD Expression

Antisense modulation of RAIDD expression can be assayed in a variety ofways known in the art. For example, RAIDD mRNA levels can be quantitatedby, e.g., Northern blot analysis, competitive polymerase chain reaction(PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR ispresently preferred. RNA analysis can be performed on total cellular RNAor poly(A)+mRNA. Methods of RNA isolation are taught in, for example,Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1,pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northernblot analysis is routine in the art and is taught in, for example,Ausubel, F. M. et al., Current Protocols in Molecular Biology, Volume 1,pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-time quantitative(PCR) can be conveniently accomplished using the commercially availableABI PRIS™ 7700 Sequence Detection System, available from PE-AppliedBiosystems, Foster City, Calif. and used according to manufacturer'sinstructions.

Protein levels of RAIDD can be quantitated in a variety of ways wellknown in the art, such as immunoprecipitation, Western blot analysis(immunoblotting), ELISA or fluorescence-activated cell sorting (FACS).Antibodies directed to RAIDD can be identified and obtained from avariety of sources, such as the MSRS catalog of antibodies (AerieCorporation, Birmingham, Mich.), or can be prepared via conventionalantibody generation methods. Methods for preparation of polyclonalantisera are taught in, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, JohnWiley & Sons, Inc., 1997. Preparation of monoclonal antibodies is taughtin, for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998.Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons,Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard inthe art and can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley& Sons, Inc., 1991.

Example 11

Poly(A)+ mRNA Isolation

Poly(A)+ mRNA was isolated according to Miura et al., Clin. Chem., 1996,42, 1758-1764. Other methods for poly(A)+mRNA isolation are taught in,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.Briefly, for cells grown on 96-well plates, growth medium was removedfrom the cells and each well was washed with 200 μL cold PBS. 60 μLlysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40,20 mM vanadyl-ribonucleoside complex) was added to each well, the platewas gently agitated and then incubated at room temperature for fiveminutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-wellplates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutesat room temperature, washed 3 times with 200 μL of wash buffer (10 mMTris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the platewas blotted on paper towels to remove excess wash buffer and thenair-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6),preheated to 70° C. was added to each well, the plate was incubated on a90° C. hot plate for 5 minutes, and the eluate was then transferred to afresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Example 12

Total RNA Isolation

Total RNA was isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 100 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 100 μL of 70% ethanol was then addedto each well and the contents mixed by pipetting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 15 seconds. 1 mL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and the vacuumagain applied for 15 seconds. 1 mL of Buffer RPE was then added to eachwell of the RNEASY 96™ plate and the vacuum applied for a period of 15seconds. The Buffer RPE wash was then repeated and the vacuum wasapplied for an additional 10 minutes. The plate was then removed fromthe QIAVAC™ manifold and blotted dry on paper towels. The plate was thenre-attached to the QIAVAC™ manifold fitted with a collection tube rackcontaining 1.2 mL collection tubes. RNA was then eluted by pipetting 60μL water into each well, incubating 1 minute, and then applying thevacuum for 30 seconds. The elution step was repeated with an additional60 μL water.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13

Real-time Quantitative PCR Analysis of RAIDD mRNA Levels

Quantitation of RAIDD mRNA levels was determined by real-timequantitative PCR using the ABI PRISM™ 7700 Sequence Detection System(PE-Applied Biosystems, Foster City, Calif.) according to manufacturer'sinstructions. This is a closed-tube, non-gel-based, fluorescencedetection system which allows high-throughput quantitation of polymerasechain reaction (PCR) products in real-time. As opposed to standard PCR,in which amplification products are quantitated after the PCR iscompleted, products in real-time quantitative PCR are quantitated asthey accumulate. This is accomplished by including in the PCR reactionan oligonucleotide probe that anneals specifically between the forwardand reverse PCR primers, and contains two fluorescent dyes. A reporterdye (e.g., JOE, FAM, or VIC, obtained from either Operon TechnologiesInc., Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either Operon Technologies Inc., Alameda, Calif. orPE-Applied Biosystems, Foster City, Calif.) is attached to the 3′ end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3′ quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5′-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular intervals by laser opticsbuilt into the ABI PRISM™ 7700 Sequence Detection System. In each assay,a series of parallel reactions containing serial dilutions of mRNA fromuntreated control samples generates a standard curve that is used toquantitate the percent inhibition after antisense oligonucleotidetreatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

PCR reagents were obtained from PE-Applied Biosystems, Foster City,Calif. RT-PCR reactions were carried out by adding 25 μL PCR cocktail(1×TAQMAN M buffer A, 5.5 mM MgCl₂, 300 μM each of DATP, dCTP and dGTP,600 μM of dUTP, 100 nM each of forward primer, reverse primer, andprobe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5Units MuLV reverse transcriptase) to 96 well plates containing 25 μLtotal RNA solution. The RT reaction was carried out by incubation for 30minutes at 48° C. Following a 10 minute incubation at 95° C. to activatethe AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol were carriedout: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5minutes (annealing/extension).

Gene target quantities obtained by real time RT-PCR are normalized usingeither the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real timeRT-PCR, by being run simultaneously with the target, multiplexing, orseparately. Total RNA is quantified using RiboGreen™ RNA quantificationreagent from Molecular Probes. Methods of RNA quantification byRiboGreen™ are taught in Jones, L. J., et al, Analytical Biochemistry,1998, 265, 368-374.

In this assay, 175 μL of RiboGreen™ working reagent (RiboGreen™ reagentdiluted 1:2865 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96L-well plate containing 25 uL purified, cellular RNA. The plate isread in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 480nm and emission at 520 nm.

Probes and primers to human RAIDD were designed to hybridize to a humanRAIDD sequence, using published sequence information (GenBank accessionnumber U79115, incorporated herein as SEQ ID NO:3). For human RAIDD thePCR primers were:

forward primer: GGGACTGTCCCAGACGGATA (SEQ ID NO: 4)

reverse primer: ACCTGCGACTGCACGTTGT (SEQ ID NO: 5) and the

PCR probe was: FAM-ACCGCTGTAAGGCCAACCACCCC-TAMRA (SEQ ID NO: 6) whereFAM (PE-Applied Biosystems, Foster City, Calif.) is the fluorescentreporter dye) and TAMRA (PE-Applied Biosystems, Foster City, Calif.) isthe quencher dye. For human GAPDH the PCR primers were:

forward primer: CAACGGATTTGGTCGTATTGG (SEQ ID NO: 7)

reverse primer: GGCAACAATATCCACTTTACCAGAGT (SEQ ID NO: 8) and

the PCR probe was: 5′ JOE-CGCCTGGTCACCAGGGCTGCT- TAMRA 3′ (SEQ ID NO: 9)where JOE (PE-Applied Biosystems, Foster City, Calif.) is thefluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City,Calif.) is the quencher dye.

Probes and primers to mouse RAIDD were designed to hybridize to a mouseRAIDD sequence, using published sequence information (GenBank accessionnumber U87229, incorporated herein as SEQ ID NO:10). For mouse RAIDD thePCR primers were:

forward primer: TGACTGGATGGCCGGAAT (SEQ ID NO:11)

reverse primer: GCTGGTTAATCTGCTGGTCTGA (SEQ ID NO: 12) and

the PCR probe was: FAM-CCTCACACATCCTCAGCAGCTCGC-TAMRA (SEQ ID NO: 13)where FAM (PE-Applied Biosystems, Foster City, Calif.) is thefluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City,Calif.) is the quencher dye. For mouse GAPDH the PCR primers were:

forward primer: GGCAAATTCAACGGCACAGT (SEQ ID NO: 14)

reverse primer: GGGTCTCGCTCCTGGAAGCT (SEQ ID NO: 15) and the

PCR probe was: 5′ JOE-AAGGCC GAGAATGGGAAGCTTGTCATC- TAMRA 3′ (SEQ ID NO:16) where JOE (PE-Applied Biosystems, Foster City, Calif.) is thefluorescent reporter dye) and TAMRA (PE-Applied Biosystems, Foster City,Calif.) is the quencher dye.

Example 14

Northern Blot Analysis of RAIDD mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by Uw visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then robedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent onditions.

To detect human RAIDD, a human RAIDD specific probe was prepared by PCRusing the forward primer GGGACTGTCCCAGACGGATA (SEQ ID NO: 4) and thereverse primer ACCTGCGACTGCACGTTGT (SEQ ID NO: 5). To normalize forvariations in loading and transfer efficiency membranes were strippedand probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH)RNA (Clontech, Palo Alto, Calif.).

To detect mouse RAIDD, a mouse RAIDD specific probe was prepared by PCRusing the forward primer TGACTGGATGGCCGGAAT (SEQ ID NO:11) and thereverse primer GCTGGTTAATCTGCTGGTCTGA (SEQ ID NO: 12). To normalize forvariations in loading and transfer efficiency membranes were strippedand probed for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH)RNA (Clontech, Palo Alto, Calif.).

Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15 Antisense Iinhibition of Human RAIDD Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

In accordance with the present invention, a series of oligonucleotideswere designed to target different regions of the human RAIDD RNA, usingpublished sequences (GenBank accession number U79115, incorporatedherein as SEQ ID NO: 3, GenBank accession number AW136381, thecomplement of which is incorporated herein as SEQ ID NO: 17, nucleotides117001-120000 of GenBank accession number AC012085, incorporated hereinas SEQ ID NO: 18, nucleotides 137001-139000 of GenBank accession numberAC023589, incorporated herein as SEQ ID NO: 19, GenBank accession numberAI417529, the complement of which is incorporated herein as SEQ ID NO:20, and GenBank accession number AA290955, incorporated herein as SEQ IDNO: 21). The oligonucleotides are shown in Table 1. “Target site”indicates the first (5′-most) nucleotide number on the particular targetsequence to which the oligonucleotide binds. All compounds in Table 1are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length,composed of a central “gap” region consisting of ten2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on human RAIDD MRNA levels by quantitative real-time PCR asdescribed in other examples herein. Data are averages from twoexperiments. If present, “N.D.” indicates “no data”.

TABLE 1 Inhibition of human RAIDD mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET TARGET ISIS # REGION SEQ ID NO SITE SEQUENCE % INHIB SEQ ID NO141760 Exon 18 4 ggagaagggctgagctgaac 58 22 141761 5′UTR 3 11aacctgccccataagcactt 81 23 141762 5′UTR 3 17 ttagggaacctgccccataa 94 24141763 5′UTR 3 21 actgttagggaacctgcccc 91 25 141764 5′UTR 3 28aatcctgactgttagggaac 82 26 141765 5′UTR 3 35 caaccggaatcctgactgtt 84 27141766 Intron 21 36 ttccaaggctgggatgcccc 61 28 141767 5′UTR 3 44gaaaaactgcaaccggaatc 86 29 141768 Intron 21 80 ccagctccctctggcactgt 7530 141769 Start 3 85 ccatttctccgtctgcaacc 67 31 Codon 141770 Start 3 93tctggcctccatttctccgt 85 32 Codon 141771 Intron 21 105gaattcaagaatggctgcac 78 33 141772 Coding 3 114 tgagcggagtacttgtttgt 8734 141773 Coding 3 122 aggcgaagtgagcggagtac 78 35 141774 Coding 3 130ccagctccaggcgaagtgag 89 36 141775 Coding 3 132 acccagctccaggcgaagtg 9237 141776 Coding 3 161 agaaccagtccctccaccaa 38 38 141777 Coding 3 164tgaagaaccagtccctccac 39 39 141778 Coding 3 170 aggtactgaagaaccagtcc 7540 141779 Coding 3 173 tagaggtactgaagaaccag 55 41 141780 Coding 3 184ttccttcctggtagaggtac 17 42 141781 Gene 19 204 ctgctttgtaacaggctttt 80 43141782 Coding 3 204 aatatggttttccgtcaaga 69 44 141783 Coding 3 220gagcattgatttcttgaata 86 45 141784 Coding 3 229 ctgtggtttgagcattgatt 6746 141785 Coding 3 232 ggcctgtggtttgagcattg 65 47 141786 Coding 3 236cggaggcctgtggtttgagc 75 48 141787 Coding 3 247 gcattgttttccggaggcct 3249 141788 Coding 3 250 ggagcattgttttccggagg 85 50 141789 Exon 18 257ctgcactccccattaaccag 33 51 141790 Coding 3 283 ctttagggcccctggaaggt 8952 141791 Coding 3 288 aaatgctttagggcccctgg 85 53 141792 Coding 3 302tctaggaatgtatcaaatgc 92 54 141793 Coding 3 312 ctgtagggaatctaggaatg 8755 141794 Coding 3 314 tcctgtagggaatctaggaa 79 56 141795 Coding 3 316actcctgtagggaatctagg 91 57 141796 Exon 17 317 taatgtgatgacatgagata 71 58141797 Coding 3 328 tgacccagggaaactcctgt 93 59 141798 Coding 3 335ttctccctgacccagggaaa 76 60 141799 Exon 17 338 tacatatgagacacctgcct 87 61141800 Coding 3 346 ccttcttcagcttctccctg 50 62 141801 Coding 3 378tgcaggcaggtcggtcatgg 0 63 141802 Coding 3 383 tcacctgcaggcaggtcggt 92 64141803 Coding 3 389 aatctgtcacctgcaggcag 79 65 141804 Exon 20 426ggtgagattgtcaacaggct 92 66 141805 Coding 3 450 cagctggttaatctgccggt 1267 141806 Exon 20 470 gcaactgcatctcattggaa 82 68 141807 Coding 3 487gcaccatgggctcccactca 85 69 141808 Coding 3 490 acagcaccatgggctcccac 7870 141809 Coding 3 507 ctgggacagtcccagagaca 45 71 141810 Coding 3 515atatccgtctgggacagtcc 75 72 141811 Coding 3 527 ttacagcggtagatatccgt 7273 141812 Coding 3 559 cctgcgactgcacgttgtgg 86 74 141813 Coding 3 562ccacctgcgactgcacgttg 84 75 141814 Coding 3 563 accacctgcgactgcacgtt 9476 141815 Coding 3 572 aaggcctccaccacctgcga 74 77 141816 Exon 20 577tggtcagtgttgctgcagtc 57 78 141817 Exon 20 587 ggatttgctttggtcagtgt 86 79141818 Coding 3 639 agcccgcagcccgttgtgca 66 80 141819 Stop 3 690gaggcaccatcactccaaca 73 81 Codon 141820 3′UTR 3 737 ggattcaagcccacatgact73 82 141821 3′UTR 3 744 aaagtcaggattcaagccca 83 83 141822 3′UTR 3 749gagtgaaagtcaggattcaa 87 84 141823 3′UTR 3 758 acctgctctgagtgaaagtc 80 85141824 3′UTR 3 843 tttcaagtaatgtctagtgg 87 86 141825 3′UTR 3 850tctggcctttcaagtaatgt 82 87 141826 3′UTR 3 862 tctgctgagtaatctggcct 72 88141827 3′UTR 3 868 gggagatctgctgagtaatc 60 89 141828 3′UTR 3 873aacatgggagatctgctgag 90 90 141829 3′UTR 3 878 gagccaacatgggagatctg 89 91141830 3′UTR 3 880 ttgagccaacatgggagatc 88 92 141831 3′UTR 3 893aaacaaagaattgttgagcc 78 93 141832 3′UTR 3 917 acaatgcaatcttcaagcaa 81 94141833 Gene 19 1168 tccccagcggttgctggagg 63 95 141834 Intron 18 1238gtcagtagccaggcacgatt 76 96 141835 Gene 19 1540 gacaacattcaggtaaatgt 8897 141836 Gene 19 1623 catgagtgaggcacattata 64 98 141837 Intron 18 2526agcagggcatggtggcaaat 90 99

As shown in Table 1, SEQ ID NOs 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 40, 41, 43, 44, 45, 46, 47, 48, 50, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 64, 65, 66, 68, 69, 70, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98 and 99 demonstrated at least 50% inhibition of humanRAIDD expression in this assay and are therefore preferred. The targetsites to which these preferred sequences are complementary are hereinreferred to as “active sites” and are therefore preferred sites fortargeting by compounds of the present invention.

Example 16

Antisense Inhibition of Mouse RAIDD Expression by ChimericPhosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

In accordance with the present invention, a second series ofoligonucleotides were designed to target different regions of the mouseRAIDD RNA, using published sequences (GenBank accession number U87229,incorporated herein as SEQ ID NO: 10). The oligonucleotides are shown inTable 2. “Target site” indicates the first (5′-most) nucleotide numberon the particular target sequence to which the oligonucleotide binds.All compounds in Table 2 are chimeric oligonucleotides (“gapmers”) 20nucleotides in length, composed of a central “gap” region consisting often 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′directions) by five-nucleotide “wings”. The wings are composed of2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone)linkages are phosphorothioate (P═S) throughout the oligonucleotide. Allcytidine residues are 5-methylcytidines. The compounds were analyzed fortheir effect on mouse RAIDD mRNA levels by quantitative real-time PCR asdescribed in other examples herein. Data are averages from twoexperiments. If present, “N.D.” indicates “no data”.

TABLE 2 Inhibition of mouse RAIDD mRNA levels by chimericphosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gapTARGET TARGET ISIS # REGION SEQ ID NO SITE SEQUENCE % INHIB SEQ ID NO134722 Start 10 1 tgcttgtctctggcttccat 85 100 Codon 134723 Start 10 2ctgcttgtctctggcttcca 81 101 Codon 134724 Coding 10 14ggagcggagtacctgcttgt 67 102 134725 Coding 10 20 acgcagggagcggagtacct 74103 134726 Coding 10 25 tccagacgcagggagcggag 61 104 134727 Coding 10 30ccagctccagacgcagggag 75 105 134728 Coding 10 35 ggcacccagctccagacgca 71106 134729 Coding 10 50 ttccaccagtacctcggcac 62 107 134730 Coding 10 101gtggttttctgtcaaaattc 79 108 134731 Coding 10 150 acagcattgtcttccggagg 84109 134732 Coding 10 154 agcaacagcattgtcttccg 66 110 134733 Coding 10159 tgtccagcaacagcattgtc 79 111 134734 Coding 10 164caggatgtccagcaacagca 77 112 134735 Coding 10 169 gaaggcaggatgtccagcaa 68113 134736 Coding 10 196 aaggtgtcaaaagctttggg 60 114 134737 Coding 10201 cgaggaaggtgtcaaaagct 73 115 134738 Coding 10 205gaatcgaggaaggtgtcaaa 55 116 134739 Coding 10 215 ttcctggagggaatcgagga 67117 134740 Coding 10 228 ttacccagggaaattcctgg 41 118 134741 Coding 10234 tctctcttacccagggaaat 66 119 134742 Coding 10 241tccagcttctctcttaccca 83 120 134743 Coding 10 246 ccttctccagcttctctctt 73121 134744 Coding 10 251 tctcgccttctccagcttct 82 122 134745 Coding 10261 tgacttcctctctcgccttc 84 123 134746 Coding 10 266ggctgtgacttcctctctcg 71 124 134747 Coding 10 271 agctcggctgtgacttcctc 81125 134748 Coding 10 276 taggcagctcggctgtgact 77 126 134749 Coding 10281 acctgtaggcagctcggctg 65 127 134750 Coding 10 287ccagtcacctgtaggcagct 53 128 134751 Coding 10 292 gccatccagtcacctgtagg 68129 134752 Coding 10 299 gattccggccatccagtcac 79 130 134753 Coding 10324 gcgagctgctgaggatgtgt 77 131 134754 Coding 10 335ctggtctgatggcgagctgc 82 132 134755 Coding 10 426 tgcagcggtagatgtcggtc 60133 134756 Coding 10 479 gcggacaaaggcctccacca 48 134 134757 Coding 10485 gcgccagcggacaaaggcct 65 135 134758 Coding 10 519agcttaggaaggtggcctgc 55 136 134759 Coding 10 525 tgtgtaagcttaggaaggtg 53137 134760 Coding 10 530 gcccttgtgtaagcttagga 43 138 134761 Coding 10535 tggaggcccttgtgtaagct 63 139 134762 Coding 10 540ctgcctggaggcccttgtgt 48 140 134763 Coding 10 545 ctccactgcctggaggccct 46141 134764 Coding 10 550 tcagcctccactgcctggag 70 142 134765 Coding 10555 agggatcagcctccactgcc 64 143 134766 Coding 10 574agcatgtgctggagcaggga 62 144 134767 Coding 10 9 ggagtacctgcttgtctctg 62145 134768 Coding 10 46 accagtacctcggcacccag 65 146 134769 Coding 10 55agtccttccaccagtacctc 79 147 134770 Coding 10 60 gaaccagtccttccaccagt 75148 134771 Coding 10 86 aattccttcctggtaaaggt 64 149 134772 Coding 10 92tgtcaaaattccttcctggt 79 150 134773 Coding 10 106 tgaatgtggttttctgtcaa 77151 134774 Coding 10 120 gagctttgatttcttgaatg 83 152 134775 Coding 10175 cccctggaaggcaggatgtc 73 153 134776 Coding 10 220ggaaattcctggagggaatc 57 154 134777 Coding 10 274 ggcagctcggctgtgacttc 74155 134778 Coding 10 321 agctgctgaggatgtgtgag 77 156 134779 Coding 10330 ctgatggcgagctgctgagg 75 157 134780 Coding 10 340atctgctggtctgatggcga 69 158 134781 Coding 10 353 agccagctggttaatctgct 70159 134782 Coding 10 358 ttctgagccagctggttaat 39 160 134783 Coding 10376 tcccactccgggcctagctt 67 161 134784 Coding 10 379ggctcccactccgggcctag 66 162 134785 Coding 10 388 aggaccacgggctcccactc 85163 134786 Coding 10 393 gagacaggaccacgggctcc 52 164 134787 Coding 10399 gtcccagagacaggaccacg 50 165 134788 Coding 10 414tgtcggtctgggacagtccc 29 166 134789 Coding 10 419 gtagatgtcggtctgggaca 63167 134790 Coding 10 432 tggccttgcagcggtagatg 66 168 134791 Coding 10437 atggttggccttgcagcggt 51 169 134792 Coding 10 475acaaaggcctccaccacctg 52 170 134793 Coding 10 507 tggcctgcttcccaaaacgc 75171 134794 Coding 10 515 taggaaggtggcctgcttcc 65 172 134795 Coding 10578 ctccagcatgtgctggagca 44 173 134796 Stop 10 581 tcactccagcatgtgctgga33 174 Codon 134797 Coding 10 363 ctagcttctgagccagctgg 34 175 134798Coding 10 368 cgggcctagcttctgagcca 66 176 134799 Coding 10 373cactccgggcctagcttctg 44 177

As shown in Table 2, SEQ ID NOs 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 167, 168, 169, 170, 171, 172, 173, 176 and 177 demonstrated atleast 35% inhibition of mouse RAIDD expression in this experiment andare therefore preferred. The target sites to which these preferredsequences are complementary are herein referred to as “active sites” andare therefore preferred sites for targeting by compounds of the presentinvention.

Example 17

Western Blot Analysis of RAIDD Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to RAIDD is used, with aradiolabelled or fluorescently labeled secondary antibody directedagainst the primary antibody species. Bands are visualized using aPHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

177 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1 tccgtcatcgctcctcaggg 20 2 20 DNA Artificial Sequence Antisense Oligonucleotide 2atgcattctg cccccaagga 20 3 949 DNA Homo sapiens CDS (101)...(700) 3ttgtgctcta aagtgcttat ggggcaggtt ccctaacagt caggattccg gttgcagttt 60ttctcccccg ccccaaagat acgtggttgc agacggagaa atg gag gcc aga gac 115 MetGlu Ala Arg Asp 1 5 aaa caa gta ctc cgc tca ctt cgc ctg gag ctg ggt gcagag gta ttg 163 Lys Gln Val Leu Arg Ser Leu Arg Leu Glu Leu Gly Ala GluVal Leu 10 15 20 gtg gag gga ctg gtt ctt cag tac ctc tac cag gaa gga atcttg acg 211 Val Glu Gly Leu Val Leu Gln Tyr Leu Tyr Gln Glu Gly Ile LeuThr 25 30 35 gaa aac cat att caa gaa atc aat gct caa acc aca ggc ctc cggaaa 259 Glu Asn His Ile Gln Glu Ile Asn Ala Gln Thr Thr Gly Leu Arg Lys40 45 50 aca atg ctc ctg ctg gat atc cta cct tcc agg ggc cct aaa gca ttt307 Thr Met Leu Leu Leu Asp Ile Leu Pro Ser Arg Gly Pro Lys Ala Phe 5560 65 gat aca ttc cta gat tcc cta cag gag ttt ccc tgg gtc agg gag aag355 Asp Thr Phe Leu Asp Ser Leu Gln Glu Phe Pro Trp Val Arg Glu Lys 7075 80 85 ctg aag aag gca agg gaa gag gcc atg acc gac ctg cct gca ggt gac403 Leu Lys Lys Ala Arg Glu Glu Ala Met Thr Asp Leu Pro Ala Gly Asp 9095 100 aga ttg act ggg atc ccc tcg cac atc ctc aac agc tcc cca tca gac451 Arg Leu Thr Gly Ile Pro Ser His Ile Leu Asn Ser Ser Pro Ser Asp 105110 115 cgg cag att aac cag ctg gcc cag agg ctg ggc cct gag tgg gag ccc499 Arg Gln Ile Asn Gln Leu Ala Gln Arg Leu Gly Pro Glu Trp Glu Pro 120125 130 atg gtg ctg tct ctg gga ctg tcc cag acg gat atc tac cgc tgt aag547 Met Val Leu Ser Leu Gly Leu Ser Gln Thr Asp Ile Tyr Arg Cys Lys 135140 145 gcc aac cac ccc cac aac gtg cag tcg cag gtg gtg gag gcc ttc atc595 Ala Asn His Pro His Asn Val Gln Ser Gln Val Val Glu Ala Phe Ile 150155 160 165 cgt tgg cgg cag cgc ttc ggg aag cag gcc acc ttc cag agc ctgcac 643 Arg Trp Arg Gln Arg Phe Gly Lys Gln Ala Thr Phe Gln Ser Leu His170 175 180 aac ggg ctg cgg gct gtg gag gtg gac ccc tcg ctg ctc ctg cacatg 691 Asn Gly Leu Arg Ala Val Glu Val Asp Pro Ser Leu Leu Leu His Met185 190 195 ttg gag tga tggtgcctcc agcaaccgct ggggagtgtg tccctgagtcatgtgggctt 750 Leu Glu 200 gaatcctgac tttcactcag agcaggtggt tttttgtgtaggtttgtttt ttatttttga 810 tgatcttcag atggaaggag aaaacagggt ttccactagacattacttga aaggccagat 870 tactcagcag atctcccatg ttggctcaac aattctttgtttttaattgc ttgaagattg 930 cattgttgta attgttcag 949 4 20 DNA ArtificialSequence PCR Primer 4 gggactgtcc cagacggata 20 5 19 DNA ArtificialSequence PCR Primer 5 acctgcgact gcacgttgt 19 6 23 DNA ArtificialSequence PCR Probe 6 accgctgtaa ggccaaccac ccc 23 7 21 DNA ArtificialSequence PCR Primer 7 caacggattt ggtcgtattg g 21 8 26 DNA ArtificialSequence PCR Primer 8 ggcaacaata tccactttac cagagt 26 9 21 DNAArtificial Sequence PCR Probe 9 cgcctggtca ccagggctgc t 21 10 600 DNAMus musculus CDS (1)...(600) 10 atg gaa gcc aga gac aag cag gta ctc cgctcc ctg cgt ctg gag ctg 48 Met Glu Ala Arg Asp Lys Gln Val Leu Arg SerLeu Arg Leu Glu Leu 1 5 10 15 ggt gcc gag gta ctg gtg gaa gga ctg gttctt cag tac ctt tac cag 96 Gly Ala Glu Val Leu Val Glu Gly Leu Val LeuGln Tyr Leu Tyr Gln 20 25 30 gaa gga att ttg aca gaa aac cac att caa gaaatc aaa gct caa acc 144 Glu Gly Ile Leu Thr Glu Asn His Ile Gln Glu IleLys Ala Gln Thr 35 40 45 aca ggc ctc cgg aag aca atg ctg ttg ctg gac atcctg cct tcc agg 192 Thr Gly Leu Arg Lys Thr Met Leu Leu Leu Asp Ile LeuPro Ser Arg 50 55 60 ggc ccc aaa gct ttt gac acc ttc ctc gat tcc ctc caggaa ttt ccc 240 Gly Pro Lys Ala Phe Asp Thr Phe Leu Asp Ser Leu Gln GluPhe Pro 65 70 75 80 tgg gta aga gag aag ctg gag aag gcg aga gag gaa gtcaca gcc gag 288 Trp Val Arg Glu Lys Leu Glu Lys Ala Arg Glu Glu Val ThrAla Glu 85 90 95 ctg cct aca ggt gac tgg atg gcc gga atc ccc tca cac atcctc agc 336 Leu Pro Thr Gly Asp Trp Met Ala Gly Ile Pro Ser His Ile LeuSer 100 105 110 agc tcg cca tca gac cag cag att aac cag ctg gct cag aagcta ggc 384 Ser Ser Pro Ser Asp Gln Gln Ile Asn Gln Leu Ala Gln Lys LeuGly 115 120 125 ccg gag tgg gag ccc gtg gtc ctg tct ctg gga ctg tcc cagacc gac 432 Pro Glu Trp Glu Pro Val Val Leu Ser Leu Gly Leu Ser Gln ThrAsp 130 135 140 atc tac cgc tgc aag gcc aac cat ccc cac aac gtg cat tcgcag gtg 480 Ile Tyr Arg Cys Lys Ala Asn His Pro His Asn Val His Ser GlnVal 145 150 155 160 gtg gag gcc ttt gtc cgc tgg cgc cag cgt ttt ggg aagcag gcc acc 528 Val Glu Ala Phe Val Arg Trp Arg Gln Arg Phe Gly Lys GlnAla Thr 165 170 175 ttc cta agc tta cac aag ggc ctc cag gca gtg gag gctgat ccc tcc 576 Phe Leu Ser Leu His Lys Gly Leu Gln Ala Val Glu Ala AspPro Ser 180 185 190 ctg ctc cag cac atg ctg gag tga 600 Leu Leu Gln HisMet Leu Glu 195 200 11 18 DNA Artificial Sequence PCR Primer 11tgactggatg gccggaat 18 12 22 DNA Artificial Sequence PCR Primer 12gctggttaat ctgctggtct ga 22 13 24 DNA Artificial Sequence PCR Probe 13cctcacacat cctcagcagc tcgc 24 14 20 DNA Artificial Sequence PCR Primer14 ggcaaattca acggcacagt 20 15 20 DNA Artificial Sequence PCR Primer 15gggtctcgct cctggaagct 20 16 27 DNA Artificial Sequence PCR Probe 16aaggccgaga atgggaagct tgtcatc 27 17 479 DNA Homo sapiens unsure 88unknown 17 ctggggagtg tgtccctgag tcatgtgggc tgaatcctga ctttcactcagagcaggtgg 60 ttttttgtgt aggtttgttt tttatttntg atgatcttca gatggaaggagaaaacaggg 120 tttccactag acattacttg aaaggccaga ttactcagca gatctcccatgttggctcaa 180 caattctttg tttttaattg cttgaagatt gcattgttgt aattgttcagtttttaaatg 240 tgtaatggca ttttaataga ctagtaaatc acagtggttc aaaatatatatccatatata 300 tatatatcca tatatatatc tcatgtcatc acattacagg caggtgtctcatatgtaaaa 360 catttacctg aatgttgtct gaggactgaa ctgtggactt tactattcataatgataaaa 420 taataaaatg cgaattacta tatataatgt gcctcactca tgaaaaaaaaaaaaaaaaa 479 18 3000 DNA Homo sapiens 18 gatgttcagc tcagcccttctccagccttt ccaatgattt cgaatgtccc caattccccc 60 taacgaatcc ctttctgactaggagtaact agctgagtgc tgcaatgaaa tcctgactga 120 cagatttgta aactctttgcgggcaggaac ccttgatata aataaatgac cttgcatttt 180 aatggaggga agcagaaggataaacaaatg aataagtgaa aagttttaat aagatggtga 240 tgcgtaaaaa ataaagctggttaatgggga gtgcaggaaa tgagcgcagc tgtcttgccg 300 ttttaatcag ataggtcagagaaggcttct ctactagggt ggcttttgac ctgcctctct 360 gttaggtcaa ttgtgtgcgaggccctgatg gagttactgt gttaaaatgc aatgattcaa 420 ttactccatt acttagataccctttaagtg acactacggt tgtccatctc cccgctattg 480 aattgtttac ccacagtaagcatggcggga agagcttcga tccgaccctt gaacgctcaa 540 taaaaatggc gacagcacttcctgtacttg ccctcaacaa agatggtgtc aacacttcct 600 gtttagggtt ttcgccattaacaaagatgg tgcttatggg gcaggttccc taacagtcag 660 gattccggtt gcagtttttctcccccgccc caaagatacg tggttgcaga cgtaagtaac 720 aggaatccat ctttctttgaaagtcctgca ttgtagcgtc tgcgacctgg gccctgcggg 780 ggcttccccg ccccgctaacttgctccgca tttccgatcc gggccaaggc tgggtggctg 840 cggcagtgcc ggaggcccgctctccccttg gtccgctctt tagttcattc ccagcactcc 900 ctctttagcg ggtcaagttgccgcagaaac cgcccgtagt cttcctgttt gatacaaggg 960 tttcagcggc cgaagcagagcctttttaaa tagttcccaa tgggcgctca cggccaaacg 1020 tgaacacctc ccctcctcacctgtcacctc ctgaacttta cagggttgcg agggggagtg 1080 tgggatcggg tgtcgcctagccgtggtcgt actgcccggg tgtgtctgaa gccccatctg 1140 tcatccctgc ctgccttcccggtcacttaa ccttcactgc cttacacaca accaagccac 1200 agcacactgg tgcaggggtggtggaactgc accttcaaat cgtgcctggc tactgaccag 1260 tgctggggtg tgtgctttttttgaaagaaa aaaatcccat aaaatgtccc atgcatgtgc 1320 ggccacgaca gtgttgtgctggagattcgg aattcttagt gggaaggaat ggagtctgtg 1380 actgtatatt gtgtaaagtttattgtagac tgggcacaaa caacaatttt cttaaaggtg 1440 atcgtgtaga ggacttttagcgaaaagagc cttaaattct gaacctgctg atttctgtcc 1500 tgcactccta ttactgatcaggttttgcaa atacttgtga tcaaaaaaag gggacctgcg 1560 tccaccttgg cagtttctgccttggtggtg gtggtgcagg tctgatgagg gcaatgtgaa 1620 aataaaacca ccatttcttctccagcgtgt taaaattagt gctgctctgt aatcttcaga 1680 taaaaattgc ttgttcagaagggtctctga cccacttttt ttttttcttt ttccaaattt 1740 ctttattgaa cgaggaattgagctgggacc ccattgtgtg aaaagcagaa acagaaaaga 1800 tgattcatct tcattgttgttcattatgtt aggaaacatt caaaattaac ctggcttggc 1860 ttaatcagtg aattaaataccatgacctgg cagtctgcta tggtttaaag atgtgcttga 1920 cctgaaggaa cttacattgcagtgacactg tctttagggc cacatcaaca tgtctgtaat 1980 ttgagctgtg attttggctctttccaggga gaaatggagg ccagagacaa acaagtactc 2040 cgctcacttc gcctggagctgggtgcagag gtattggtgg agggactggt tcttcagtac 2100 ctctaccagg aaggaatcttgacggaaaac catattcaag aaatcaatgc tcaaaccaca 2160 ggcctccgga aaacaatgctcctgctggat atcctacctt ccaggggccc taaagcattt 2220 gatacattcc tagattccctacaggagttt ccctgggtca gggagaagct gaagaaggca 2280 agggaagagg ccatgaccgacctgcctgca ggtaggcctc agaaagatca ctttgaacca 2340 gctccaaaat gttgtaactatgctctttgc ttttttgttt atttgtttgt ttgtttttga 2400 gacagagtct tgctctgttgcccaggctgg agtgcaatgg tgtgatctcg gctcactgca 2460 acctctgctt cctgggttcaagcaattcac ttgcttcagc ctcccgagtt gctgggaata 2520 caggcatttg ccaccatgccctgctaagtt ttgtattttt agtagagatg gggtttcatc 2580 atgttggcca ggctggtctcgaactccgta cctcaggtga tccacccacc tccgcctccc 2640 aaagtgctgg gattacagacgtgagccacc acacctagcc agaactatgc cctttggatt 2700 tgaagttggg atgtaaggcaggacaagggt caatggttga tgaaagtggg taaggatttg 2760 ggactgaagg gacatgacgttggaggaata ggatcaggag gtggcattag gaagtgcagg 2820 gaaggaagaa tgaaccgtgactgggaggtg agggagggag ctcagagaat gcatttagaa 2880 atgcttccag gaaacctggcatgtcacttg ggaagcatgt tctcatcaga gagccaggtc 2940 atatctcagc cgttggcatcaggagccatt tggaaagtgc agatgggaaa gcaggcattc 3000 19 2000 DNA Homosapiens 19 acctcatcgg cgagggtagc tgtttggttt agcctttgtc ctctagcacgtgcctttcct 60 gcagggaggg gcattcccag ccttggaaca ggccccatca ctaaggccacagacagtgcc 120 agagggagct ggggtctgtg cagccattct tgaattcccc cagaatgccttgcctggcct 180 tgagtaaaag ggatagaagt gaaaaaagcc tgttacaaag cagcatgtacgaggtaaccc 240 ttcatgtgtg ttttatatat caaagaaaat agactagtta ttatcaatgagtttttctgg 300 tgggatttct agttattttt attttgttgc tttcaaagtt tctgtaacaggcagggtgct 360 gtggctcacg cctgtaatcc tagcactttg ggaggccaag gcaggtggatcacctgaggt 420 caggagttca agaccagcct ggccaacatg gtgaaaccct gtctctgctaaaaatacaaa 480 aattagctgg gcatggtggc agttgcctgt aatcccagct acttgggaggctgaaacagg 540 agaatcactt caacccagga ggtggagttt gtagtgagcc aacatcatgccattgctctc 600 cagcctgggt gacagagtga aacaccgtct caaaaaaaaa aaaaaaaaaagtttctgtaa 660 aaacattttc acagatactt aacagcattg aagccctgaa tcatggcgttgtacagagca 720 gcccccagga agaagacaag agccaattgg ctctgccatt tggaagcattgccttcttct 780 ccccacgatt ctcatttctg cccccaaatg atgtgtttgt tgccttgctcatttcaccgg 840 ggtgtctttt tcctcctcag gtgacagatt gactgggatc ccctcgcacatcctcaacag 900 ctccccatca gaccggcaga ttaaccagct ggcccagagg ctgggccctgagtgggagcc 960 catggtgctg tctctgggac tgtcccagac ggatatctac cgctgtaaggccaaccaccc 1020 ccacaacgtg cagtcgcagg tggtggaggc cttcatccgt tggcggcagcgcttcgggaa 1080 gcaggccacc ttccagagcc tgcacaacgg gctgcgggct gtggaggtggacccctcgct 1140 gctcctgcac atgttggagt gatggtgcct ccagcaaccg ctggggagtgtgtccctgag 1200 tcatgtgggc tgaatcctga ctttcactca gagcaggtgg ttttttgtgtaggtttgttt 1260 tttatttttg atgatcttca gatggaagga gaaaacaggg tttccactagacattacttg 1320 aaaggccaga ttactcagca gatctcccat gttggctcaa caattctttgtttttaattg 1380 cttgaagatt gcattgttgt aattgttcag tttttaaatg tgtaatggcattttaataga 1440 ctagtaaatc acagtggttc aaaatatata tccatatata tatatatccatatatatatc 1500 tcatgtcatc acattacagg caggtgtctc atatgtaaaa catttacctgaatgttgtct 1560 gaggactgaa ctgtggactt tactattcat aatgataaaa taataaaatgcgaattacta 1620 tatataatgt gcctcactca tgagaaagta ccgtgttggg tttttttttttctttctcat 1680 tcctgtgttg catagattaa gggtgtaaaa ttacaatagt cttttttttctttttggaac 1740 agaattaata gtaacagtga aatggttact ttcccaagtc aggataaatagctcaggcag 1800 gatgtctgta ttaaatattg taagacacta aagctccctg caaagaccagttcaaggacc 1860 agcatgcata cccgagccct tcatctttaa aagatcgctc gagaaaatcctgtgctgttt 1920 attgaaagca ttaagtcagg ggacgagagg gggtgtggtc tttccattgaagccagaatg 1980 actgaagtgt tgtgctccta 2000 20 733 DNA Homo sapiensunsure 152 unknown 20 gaacaacaag tactccgctc cacctgcctg ggagtgggtgcaaaggtatg gtgaagggat 60 ggttcttcag tactctaccc agaaaggaat cttgacggaaaccatattca agaaatcaat 120 gctcaaacac aggcctccgg aaacaatgct cntgctggatatcctacctt ccaggggccc 180 taaagcattt gatacatcct agattcccta caggagtttccctgggtcag ggagaagctg 240 aagaaggcaa gggaagaggc catgaccgac ctgcctgcagggttagagga aaaaggaagg 300 actggccgaa gaatggggtg gggagctgga gaagagaagggccaaaagtg tcagactgtg 360 gggatgagga cctgaaggag gcccatgaga cctcaggcatgggccctgga actctgcctt 420 tttgaagcct gttgacaatc tcaccctaat ttaaccctttgtggaattct tccaatgaga 480 tgcagttgcc atcatcaggc ttcctaaaga attgagcctgcccactctgt tatgaagtct 540 acccagaaga gctggcagag taggcccctt cactgggactgcagcaacac tgaccaaagc 600 aaatccttct cttggaagaa acaaactcat tcctcacacctgcctcaccc cgctcccacc 660 gccaccccct gcaccacacg ccagccctta ataaaaactaaaactgatgt cattgtaaaa 720 aaaaaaaaaa aaa 733 21 481 DNA Homo sapiens 21cgtttgtcct ctagcacgtg cctttcctgc agggaggggc atcccagcct tggaacaggc 60cccatcacta aggccacaga cagtgccaga gggagctggg gtctgtgcag ccattcttga 120attcccccag aatgccttgc ctggccttga gtaaaaggga tagaagtgaa aaaagcctgt 180tacaaagcag catgtacgag gtgacagatt gactgggatc ccctcgcaca tcctcaacag 240ctccccatca gaccggcaga ttaaccagct ggcccagagc tgggcctgag tgggagccca 300tggtgctgtc tctgggactg tcccagacgg atatctaccg ctgtaaggcc aaccaccccc 360acaacgtgca gtcgcaggtg gtggaggcct tcatccgttg gcggcacgct tcgggaagca 420ggccaccttc cagagcctgc acaacgggct gcgggctgtg gaggtggacc ctcgctgctc 480 c481 22 20 DNA Artificial Sequence Antisense Oligonucleotide 22ggagaagggc tgagctgaac 20 23 20 DNA Artificial Sequence AntisenseOligonucleotide 23 aacctgcccc ataagcactt 20 24 20 DNA ArtificialSequence Antisense Oligonucleotide 24 ttagggaacc tgccccataa 20 25 20 DNAArtificial Sequence Antisense Oligonucleotide 25 actgttaggg aacctgcccc20 26 20 DNA Artificial Sequence Antisense Oligonucleotide 26 aatcctgactgttagggaac 20 27 20 DNA Artificial Sequence Antisense Oligonucleotide 27caaccggaat cctgactgtt 20 28 20 DNA Artificial Sequence AntisenseOligonucleotide 28 ttccaaggct gggatgcccc 20 29 20 DNA ArtificialSequence Antisense Oligonucleotide 29 gaaaaactgc aaccggaatc 20 30 20 DNAArtificial Sequence Antisense Oligonucleotide 30 ccagctccct ctggcactgt20 31 20 DNA Artificial Sequence Antisense Oligonucleotide 31 ccatttctccgtctgcaacc 20 32 20 DNA Artificial Sequence Antisense Oligonucleotide 32tctggcctcc atttctccgt 20 33 20 DNA Artificial Sequence AntisenseOligonucleotide 33 gaattcaaga atggctgcac 20 34 20 DNA ArtificialSequence Antisense Oligonucleotide 34 tgagcggagt acttgtttgt 20 35 20 DNAArtificial Sequence Antisense Oligonucleotide 35 aggcgaagtg agcggagtac20 36 20 DNA Artificial Sequence Antisense Oligonucleotide 36 ccagctccaggcgaagtgag 20 37 20 DNA Artificial Sequence Antisense Oligonucleotide 37acccagctcc aggcgaagtg 20 38 20 DNA Artificial Sequence AntisenseOligonucleotide 38 agaaccagtc cctccaccaa 20 39 20 DNA ArtificialSequence Antisense Oligonucleotide 39 tgaagaacca gtccctccac 20 40 20 DNAArtificial Sequence Antisense Oligonucleotide 40 aggtactgaa gaaccagtcc20 41 20 DNA Artificial Sequence Antisense Oligonucleotide 41 tagaggtactgaagaaccag 20 42 20 DNA Artificial Sequence Antisense Oligonucleotide 42ttccttcctg gtagaggtac 20 43 20 DNA Artificial Sequence AntisenseOligonucleotide 43 ctgctttgta acaggctttt 20 44 20 DNA ArtificialSequence Antisense Oligonucleotide 44 aatatggttt tccgtcaaga 20 45 20 DNAArtificial Sequence Antisense Oligonucleotide 45 gagcattgat ttcttgaata20 46 20 DNA Artificial Sequence Antisense Oligonucleotide 46 ctgtggtttgagcattgatt 20 47 20 DNA Artificial Sequence Antisense Oligonucleotide 47ggcctgtggt ttgagcattg 20 48 20 DNA Artificial Sequence AntisenseOligonucleotide 48 cggaggcctg tggtttgagc 20 49 20 DNA ArtificialSequence Antisense Oligonucleotide 49 gcattgtttt ccggaggcct 20 50 20 DNAArtificial Sequence Antisense Oligonucleotide 50 ggagcattgt tttccggagg20 51 20 DNA Artificial Sequence Antisense Oligonucleotide 51 ctgcactccccattaaccag 20 52 20 DNA Artificial Sequence Antisense Oligonucleotide 52ctttagggcc cctggaaggt 20 53 20 DNA Artificial Sequence AntisenseOligonucleotide 53 aaatgcttta gggcccctgg 20 54 20 DNA ArtificialSequence Antisense Oligonucleotide 54 tctaggaatg tatcaaatgc 20 55 20 DNAArtificial Sequence Antisense Oligonucleotide 55 ctgtagggaa tctaggaatg20 56 20 DNA Artificial Sequence Antisense Oligonucleotide 56 tcctgtagggaatctaggaa 20 57 20 DNA Artificial Sequence Antisense Oligonucleotide 57actcctgtag ggaatctagg 20 58 20 DNA Artificial Sequence AntisenseOligonucleotide 58 taatgtgatg acatgagata 20 59 20 DNA ArtificialSequence Antisense Oligonucleotide 59 tgacccaggg aaactcctgt 20 60 20 DNAArtificial Sequence Antisense Oligonucleotide 60 ttctccctga cccagggaaa20 61 20 DNA Artificial Sequence Antisense Oligonucleotide 61 tacatatgagacacctgcct 20 62 20 DNA Artificial Sequence Antisense Oligonucleotide 62ccttcttcag cttctccctg 20 63 20 DNA Artificial Sequence AntisenseOligonucleotide 63 tgcaggcagg tcggtcatgg 20 64 20 DNA ArtificialSequence Antisense Oligonucleotide 64 tcacctgcag gcaggtcggt 20 65 20 DNAArtificial Sequence Antisense Oligonucleotide 65 aatctgtcac ctgcaggcag20 66 20 DNA Artificial Sequence Antisense Oligonucleotide 66 ggtgagattgtcaacaggct 20 67 20 DNA Artificial Sequence Antisense Oligonucleotide 67cagctggtta atctgccggt 20 68 20 DNA Artificial Sequence AntisenseOligonucleotide 68 gcaactgcat ctcattggaa 20 69 20 DNA ArtificialSequence Antisense Oligonucleotide 69 gcaccatggg ctcccactca 20 70 20 DNAArtificial Sequence Antisense Oligonucleotide 70 acagcaccat gggctcccac20 71 20 DNA Artificial Sequence Antisense Oligonucleotide 71 ctgggacagtcccagagaca 20 72 20 DNA Artificial Sequence Antisense Oligonucleotide 72atatccgtct gggacagtcc 20 73 20 DNA Artificial Sequence AntisenseOligonucleotide 73 ttacagcggt agatatccgt 20 74 20 DNA ArtificialSequence Antisense Oligonucleotide 74 cctgcgactg cacgttgtgg 20 75 20 DNAArtificial Sequence Antisense Oligonucleotide 75 ccacctgcga ctgcacgttg20 76 20 DNA Artificial Sequence Antisense Oligonucleotide 76 accacctgcgactgcacgtt 20 77 20 DNA Artificial Sequence Antisense Oligonucleotide 77aaggcctcca ccacctgcga 20 78 20 DNA Artificial Sequence AntisenseOligonucleotide 78 tggtcagtgt tgctgcagtc 20 79 20 DNA ArtificialSequence Antisense Oligonucleotide 79 ggatttgctt tggtcagtgt 20 80 20 DNAArtificial Sequence Antisense Oligonucleotide 80 agcccgcagc ccgttgtgca20 81 20 DNA Artificial Sequence Antisense Oligonucleotide 81 gaggcaccatcactccaaca 20 82 20 DNA Artificial Sequence Antisense Oligonucleotide 82ggattcaagc ccacatgact 20 83 20 DNA Artificial Sequence AntisenseOligonucleotide 83 aaagtcagga ttcaagccca 20 84 20 DNA ArtificialSequence Antisense Oligonucleotide 84 gagtgaaagt caggattcaa 20 85 20 DNAArtificial Sequence Antisense Oligonucleotide 85 acctgctctg agtgaaagtc20 86 20 DNA Artificial Sequence Antisense Oligonucleotide 86 tttcaagtaatgtctagtgg 20 87 20 DNA Artificial Sequence Antisense Oligonucleotide 87tctggccttt caagtaatgt 20 88 20 DNA Artificial Sequence AntisenseOligonucleotide 88 tctgctgagt aatctggcct 20 89 20 DNA ArtificialSequence Antisense Oligonucleotide 89 gggagatctg ctgagtaatc 20 90 20 DNAArtificial Sequence Antisense Oligonucleotide 90 aacatgggag atctgctgag20 91 20 DNA Artificial Sequence Antisense Oligonucleotide 91 gagccaacatgggagatctg 20 92 20 DNA Artificial Sequence Antisense Oligonucleotide 92ttgagccaac atgggagatc 20 93 20 DNA Artificial Sequence AntisenseOligonucleotide 93 aaacaaagaa ttgttgagcc 20 94 20 DNA ArtificialSequence Antisense Oligonucleotide 94 acaatgcaat cttcaagcaa 20 95 20 DNAArtificial Sequence Antisense Oligonucleotide 95 tccccagcgg ttgctggagg20 96 20 DNA Artificial Sequence Antisense Oligonucleotide 96 gtcagtagccaggcacgatt 20 97 20 DNA Artificial Sequence Antisense Oligonucleotide 97gacaacattc aggtaaatgt 20 98 20 DNA Artificial Sequence AntisenseOligonucleotide 98 catgagtgag gcacattata 20 99 20 DNA ArtificialSequence Antisense Oligonucleotide 99 agcagggcat ggtggcaaat 20 100 20DNA Artificial Sequence Antisense Oligonucleotide 100 tgcttgtctctggcttccat 20 101 20 DNA Artificial Sequence Antisense Oligonucleotide101 ctgcttgtct ctggcttcca 20 102 20 DNA Artificial Sequence AntisenseOligonucleotide 102 ggagcggagt acctgcttgt 20 103 20 DNA ArtificialSequence Antisense Oligonucleotide 103 acgcagggag cggagtacct 20 104 20DNA Artificial Sequence Antisense Oligonucleotide 104 tccagacgcagggagcggag 20 105 20 DNA Artificial Sequence Antisense Oligonucleotide105 ccagctccag acgcagggag 20 106 20 DNA Artificial Sequence AntisenseOligonucleotide 106 ggcacccagc tccagacgca 20 107 20 DNA ArtificialSequence Antisense Oligonucleotide 107 ttccaccagt acctcggcac 20 108 20DNA Artificial Sequence Antisense Oligonucleotide 108 gtggttttctgtcaaaattc 20 109 20 DNA Artificial Sequence Antisense Oligonucleotide109 acagcattgt cttccggagg 20 110 20 DNA Artificial Sequence AntisenseOligonucleotide 110 agcaacagca ttgtcttccg 20 111 20 DNA ArtificialSequence Antisense Oligonucleotide 111 tgtccagcaa cagcattgtc 20 112 20DNA Artificial Sequence Antisense Oligonucleotide 112 caggatgtccagcaacagca 20 113 20 DNA Artificial Sequence Antisense Oligonucleotide113 gaaggcagga tgtccagcaa 20 114 20 DNA Artificial Sequence AntisenseOligonucleotide 114 aaggtgtcaa aagctttggg 20 115 20 DNA ArtificialSequence Antisense Oligonucleotide 115 cgaggaaggt gtcaaaagct 20 116 20DNA Artificial Sequence Antisense Oligonucleotide 116 gaatcgaggaaggtgtcaaa 20 117 20 DNA Artificial Sequence Antisense Oligonucleotide117 ttcctggagg gaatcgagga 20 118 20 DNA Artificial Sequence AntisenseOligonucleotide 118 ttacccaggg aaattcctgg 20 119 20 DNA ArtificialSequence Antisense Oligonucleotide 119 tctctcttac ccagggaaat 20 120 20DNA Artificial Sequence Antisense Oligonucleotide 120 tccagcttctctcttaccca 20 121 20 DNA Artificial Sequence Antisense Oligonucleotide121 ccttctccag cttctctctt 20 122 20 DNA Artificial Sequence AntisenseOligonucleotide 122 tctcgccttc tccagcttct 20 123 20 DNA ArtificialSequence Antisense Oligonucleotide 123 tgacttcctc tctcgccttc 20 124 20DNA Artificial Sequence Antisense Oligonucleotide 124 ggctgtgacttcctctctcg 20 125 20 DNA Artificial Sequence Antisense Oligonucleotide125 agctcggctg tgacttcctc 20 126 20 DNA Artificial Sequence AntisenseOligonucleotide 126 taggcagctc ggctgtgact 20 127 20 DNA ArtificialSequence Antisense Oligonucleotide 127 acctgtaggc agctcggctg 20 128 20DNA Artificial Sequence Antisense Oligonucleotide 128 ccagtcacctgtaggcagct 20 129 20 DNA Artificial Sequence Antisense Oligonucleotide129 gccatccagt cacctgtagg 20 130 20 DNA Artificial Sequence AntisenseOligonucleotide 130 gattccggcc atccagtcac 20 131 20 DNA ArtificialSequence Antisense Oligonucleotide 131 gcgagctgct gaggatgtgt 20 132 20DNA Artificial Sequence Antisense Oligonucleotide 132 ctggtctgatggcgagctgc 20 133 20 DNA Artificial Sequence Antisense Oligonucleotide133 tgcagcggta gatgtcggtc 20 134 20 DNA Artificial Sequence AntisenseOligonucleotide 134 gcggacaaag gcctccacca 20 135 20 DNA ArtificialSequence Antisense Oligonucleotide 135 gcgccagcgg acaaaggcct 20 136 20DNA Artificial Sequence Antisense Oligonucleotide 136 agcttaggaaggtggcctgc 20 137 20 DNA Artificial Sequence Antisense Oligonucleotide137 tgtgtaagct taggaaggtg 20 138 20 DNA Artificial Sequence AntisenseOligonucleotide 138 gcccttgtgt aagcttagga 20 139 20 DNA ArtificialSequence Antisense Oligonucleotide 139 tggaggccct tgtgtaagct 20 140 20DNA Artificial Sequence Antisense Oligonucleotide 140 ctgcctggaggcccttgtgt 20 141 20 DNA Artificial Sequence Antisense Oligonucleotide141 ctccactgcc tggaggccct 20 142 20 DNA Artificial Sequence AntisenseOligonucleotide 142 tcagcctcca ctgcctggag 20 143 20 DNA ArtificialSequence Antisense Oligonucleotide 143 agggatcagc ctccactgcc 20 144 20DNA Artificial Sequence Antisense Oligonucleotide 144 agcatgtgctggagcaggga 20 145 20 DNA Artificial Sequence Antisense Oligonucleotide145 ggagtacctg cttgtctctg 20 146 20 DNA Artificial Sequence AntisenseOligonucleotide 146 accagtacct cggcacccag 20 147 20 DNA ArtificialSequence Antisense Oligonucleotide 147 agtccttcca ccagtacctc 20 148 20DNA Artificial Sequence Antisense Oligonucleotide 148 gaaccagtccttccaccagt 20 149 20 DNA Artificial Sequence Antisense Oligonucleotide149 aattccttcc tggtaaaggt 20 150 20 DNA Artificial Sequence AntisenseOligonucleotide 150 tgtcaaaatt ccttcctggt 20 151 20 DNA ArtificialSequence Antisense Oligonucleotide 151 tgaatgtggt tttctgtcaa 20 152 20DNA Artificial Sequence Antisense Oligonucleotide 152 gagctttgatttcttgaatg 20 153 20 DNA Artificial Sequence Antisense Oligonucleotide153 cccctggaag gcaggatgtc 20 154 20 DNA Artificial Sequence AntisenseOligonucleotide 154 ggaaattcct ggagggaatc 20 155 20 DNA ArtificialSequence Antisense Oligonucleotide 155 ggcagctcgg ctgtgacttc 20 156 20DNA Artificial Sequence Antisense Oligonucleotide 156 agctgctgaggatgtgtgag 20 157 20 DNA Artificial Sequence Antisense Oligonucleotide157 ctgatggcga gctgctgagg 20 158 20 DNA Artificial Sequence AntisenseOligonucleotide 158 atctgctggt ctgatggcga 20 159 20 DNA ArtificialSequence Antisense Oligonucleotide 159 agccagctgg ttaatctgct 20 160 20DNA Artificial Sequence Antisense Oligonucleotide 160 ttctgagccagctggttaat 20 161 20 DNA Artificial Sequence Antisense Oligonucleotide161 tcccactccg ggcctagctt 20 162 20 DNA Artificial Sequence AntisenseOligonucleotide 162 ggctcccact ccgggcctag 20 163 20 DNA ArtificialSequence Antisense Oligonucleotide 163 aggaccacgg gctcccactc 20 164 20DNA Artificial Sequence Antisense Oligonucleotide 164 gagacaggaccacgggctcc 20 165 20 DNA Artificial Sequence Antisense Oligonucleotide165 gtcccagaga caggaccacg 20 166 20 DNA Artificial Sequence AntisenseOligonucleotide 166 tgtcggtctg ggacagtccc 20 167 20 DNA ArtificialSequence Antisense Oligonucleotide 167 gtagatgtcg gtctgggaca 20 168 20DNA Artificial Sequence Antisense Oligonucleotide 168 tggccttgcagcggtagatg 20 169 20 DNA Artificial Sequence Antisense Oligonucleotide169 atggttggcc ttgcagcggt 20 170 20 DNA Artificial Sequence AntisenseOligonucleotide 170 acaaaggcct ccaccacctg 20 171 20 DNA ArtificialSequence Antisense Oligonucleotide 171 tggcctgctt cccaaaacgc 20 172 20DNA Artificial Sequence Antisense Oligonucleotide 172 taggaaggtggcctgcttcc 20 173 20 DNA Artificial Sequence Antisense Oligonucleotide173 ctccagcatg tgctggagca 20 174 20 DNA Artificial Sequence AntisenseOligonucleotide 174 tcactccagc atgtgctgga 20 175 20 DNA ArtificialSequence Antisense Oligonucleotide 175 ctagcttctg agccagctgg 20 176 20DNA Artificial Sequence Antisense Oligonucleotide 176 cgggcctagcttctgagcca 20 177 20 DNA Artificial Sequence Antisense Oligonucleotide177 cactccgggc ctagcttctg 20

What is claimed is:
 1. A compound up to 50 nucleobases in lengthcomprising at least an 8-nucleobase portion of SEQ ID NO: 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 40, 41, 43, 44, 45,46, 47, 48, 50, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 64, 65, 66,68, 69, 70, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,160, 161, 162, 163, 164, 165, 167, 168, 169, 170, 171, 172, 173, 176 or177 which compound specifically hybridizes with a nucleic acid encodingRAIDD and inhibits the expression of RAIDD.
 2. The compound of claim 1which is an antisense oligonucleotide.
 3. The compound of claim 2wherein the antisense oligonucleotide comprises at least one modifiedinternucleoside linkage.
 4. The compound of claim 3 wherein the modifiedinternucleoside linkage is a phosphorothioate linkage.
 5. The compoundof claim 2 wherein the antisense oligonucleotide comprises at least onemodified sugar moiety.
 6. The compound of claim 4 wherein the modifiedsugar moiety is a 2′-O-methoxyethyl sugar moiety.
 7. The compound ofclaim 2 wherein the antisense oligonucleotide comprises at least onemodified nucleobase.
 8. The compound of claim 7 wherein the modifiednucleobase is a 5-methylcytosine.
 9. The compound of claim 2 wherein theantisense oligonucleotide is a chimeric oligonucleotide.
 10. A method ofinhibiting the expression of RAIDD in human cells or tissues comprisingcontacting said cells or tissues in vitro with the compound of claim 1so that expression of RAIDD is inhibited.
 11. A composition comprisingthe compound of claim 1 and a pharmaceutically acceptable carrier ordiluent.
 12. The composition of claim 11 further comprising a colloidaldispersion system.
 13. The composition of claim 11 wherein the compoundis an antisense oligonucleotide.